WO2021202599A2

WO2021202599A2 - Adeno-associated virus based compositions and related methods for inducing humoral immunity

Info

Publication number: WO2021202599A2
Application number: PCT/US2021/024983
Authority: WO
Inventors: Philip R. Johnson
Original assignee: Valiant Biosciences, Llc
Priority date: 2020-04-01
Filing date: 2021-03-30
Publication date: 2021-10-07
Also published as: WO2021202599A3

Abstract

Several embodiments of the methods and compositions disclosed herein relate to compositions and methods of immunizing patients against viral infections, such as those caused by coronaviruses. In some embodiments, a viral vector, such as AAV, is delivered to a subject to enable the production of an antigenic viral protein or protein fragment by the subject, in order to allow development of a humoral immune response by the subject. In some embodiments, the compositions and methods serve to vaccinate the subject against a viral infection, such as COVID-19, which is caused by SARS-CoV-2.

Description

ADENO-ASSOCIATED VIRUS BASED COMPOSITIONS AND RELATED METHODS FOR INDUCING

HUMORAL IMMUNITY

RELATED APPLICATIONS

[0001] This application claims priority to United State Provisional Patent Application No. 63/003,491 , filed April 1 , 2020, the entirety of which is incorporated by reference herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith: File Name: VLNT002WO_ST25.txt; created March 25, 2021 , 74.5 KB in size.

FIELD

[0003] Several embodiments disclosed herein relate to methods and compositions for viral-based vaccines to induce humoral immunity for infectious diseases, such as viral infections, including COVID-19 or other coronavirus-based infections. In several embodiments, the viral-based vaccine is an adeno- associated virus that encodes an immunogenic viral protein, or derivative thereof.

BACKGROUND

[0004] Infections, particularly viral infections, have the potential to run rampant due to the ability of the virus to be spread prior to a subject exhibiting symptoms of the infection. Once symptoms are evident, the subject may have already come into contact with, and thus spread, to virus to other individuals, or in some embodiments, onto surfaces. Particularly susceptible are patients with underlying conditions that make combating a viral infection more challenging. These include, but are not limited to, patients with underlying respiratory disease, immunocompromised patients or even those with a disease that affects an organ system that is adversely affected by viral infection, such as the cardiovascular system. Further, elderly patients may be at higher risk. The SARS-Cov2 virus is one such virus, and causes COVID-19. COVID-19 patients can develop symptoms, including, but not limited to cytokine storm, lymphocytopenia, acute respiratory distress syndrome, and various cardiac disease manifestations including myocarditis, myocardial infarction and arrhythmias.

[0005] Even with the early stages of this infection, mortality in patients at the highest risk (elderly, with comorbidities including prior cardiorespiratory dysfunction) approaches 45-50%, even with state-of the art supportive care in an ICU. Currently, no treatment modality has been shown to reduce mortality and morbidity in critically-ill Covid patients. Given the pandemic nature of the illness, and its high morbidity and mortality, there is a compelling and urgent unmet medical need. SUMMARY

[0006] In view of the pressing need for therapies and vaccines to treat or prevent COVID-19, or other viral infections, provided for herein are compositions, methods, and uses of engineered viral vectors for vaccination against viral pathogens. In several embodiments, there is provided a method for inducing an immune response in a host comprising administering to the host an effective amount of an infectious, engineered viral vector encoding at least a an antigenic portion of a pathogenic viral protein, thereby inducing expression of the antigenic portion of the pathogenic viral protein by the recipient subject and inducing a humoral immune response to the antigenic portion of the pathogenic viral protein. In several embodiments, the viral vector is a recombinant adeno-associated virus (rAVV). In several embodiments, the viral vector encodes at least a portion of a coronavirus spike glycoprotein. In some embodiments, the portion of the coronavirus spike glycoprotein does not comprise a complete transmembrane domain. In several embodiments, the immune response induced is a humoral immune response to the at least a portion of the coronavirus spike glycoprotein.

[0007] In several embodiments, the at least a portion of the coronavirus spike glycoprotein is at least a portion of a SARS-Cov-2 polypeptide. In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises an S1 subunit of the SARS-Cov-2 spike glycoprotein. In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises an N-terminal domain of an S1 subunit of the SARS-Cov-2 spike glycoprotein. In additional embodiments, the at least a portion of the coronavirus spike glycoprotein comprises an C-terminal domain of an S1 subunit of the SARS-Cov- 2 spike glycoprotein. In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises an S2 subunit of the SARS-Cov-2 spike glycoprotein.

[0008] In additional embodiments, the at least a portion of the coronavirus spike glycoprotein is at least a portion of a MERS-Cov-2 polypeptide. In additional embodiments, the at least a portion of the coronavirus spike glycoprotein is at least a portion of a SARS-CoV polypeptide.

[0009] In several embodiments, the lack of a complete transmembrane domain results in expression of the at least a portion of the coronavirus spike glycoprotein as a soluble protein.

[0010] In several embodiments, the at least a portion of the coronavirus spike glycoprotein does not comprise SEQ ID NO: 5.

[0011] In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In several embodiments, the inclusion of such a sequence sharing the recited identity to SEQ ID NO: 6 allows the at least a portion of the coronavirus spike glycoprotein to be expressed as a soluble trimer.

[0012] In several embodiments, the portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0013] In several embodiments, the viral vector comprises a rAAV and the rAAV has a sequence sharing at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In several embodiments, the portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to one or more of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In several embodiments, the rAAV is a serotype 1 , rh10, rh74 or is pseudotyped to serotype 1 , rh10, rh74. In several embodiments, the rAAV comprises a promoter to drive expression of the at least a portion of the coronavirus spike glycoprotein. In several embodiments, the promoter is a cytomegalovirus promoter.

[0014] In several embodiments, the rAAV is produced by a mammalian host cell stably transfected with a recombinant adeno-associated virus genome lacking adeno-associated virus rep-cap genes and stably transfected with adeno-associated virus rep-cap genes.

[0015] In several embodiments, the rAAV encoding at least a portion of a coronavirus spike glycoprotein is administered by at least one intramuscular injection.

[0016] In several embodiments, there is provided a polynucleotide encoding a first nucleic acid vector containing a first heterologous nucleic acid region encoding a first protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region, wherein the heterologous nucleic acid region encodes at least a portion of a coronavirus spike glycoprotein, but does not encode a functional transmembrane domain. In several embodiments, the at least a portion of the coronavirus spike glycoprotein does not comprise SEQ ID NO: 5. In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In several embodiments, the inclusion of such a sequence sharing the recited identity to SEQ ID NO: 6 allows the at least a portion of the coronavirus spike glycoprotein to be expressed as a soluble trimer. In several embodiments, the portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0017] In several embodiments, the lack of a functional transmembrane domain results in expression of the at least a portion of the coronavirus spike glycoprotein as a soluble protein.

[0018] In several embodiments, the polynucleotide encodes a rAAV serotype 1 , rh10, or rh74 vector.

[0019] In several embodiments, the polynucleotide encodes a rAAV with a sequence sharing at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2.

[0020] Provided for herein, in several embodiments, is a rAAV vector encoding at least a portion of a coronavirus spike glycoprotein, in several embodiments, as a soluble protein (while in other embodiments the protein is membrane-bound). In several embodiments, the vector comprises a first heterologous nucleic acid region encoding a first protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region, wherein the heterologous nucleic acid region encodes at least a portion of a coronavirus spike glycoprotein. In several embodiments, the vector does not encode a functional transmembrane domain of the coronavirus spike glycoprotein. In several embodiments, the at least a portion of the coronavirus spike glycoprotein does not comprise SEQ ID NO: 5. In several embodiments, the at least a portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In several embodiments, the inclusion of such a sequence sharing the recited identity to SEQ ID NO: 6 allows the at least a portion of the coronavirus spike glycoprotein to be expressed as a soluble trimer. In several embodiments, the portion of the coronavirus spike glycoprotein comprises a sequence having at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In several embodiments, the rAAV vector is serotype 1 , rh10, or rh74.

[0021] Also provided for herein are uses of the recombinant viral vectors disclosed herein for inducing immunity to a coronavirus infection and/or for the production of a medicament for inducing immunity to a coronavirus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows a schematic representation of a plasmid map of pTR-SARS2S-2P, discussed in further detail below.

[0023] Figure 2 shows a schematic representation of a plasmid map of pTR-SARS2S-2P-dTM, discussed in further detail below.

[0024] Figure 3 shows a schematic of AAV-CMV-SARS2 with AAV2 ITRs and pseudotyped to

AAV1 .

[0025] Figure 4 shows data related to assessment of delivery of various AAV serotypes based on an activity assay using tissue homogenates.

[0026] Figure 5 shows data related to assessment of biodistribution of various AAV serotypes to target tissue when delivered systemically.

[0027] Figures 6A-6C show data related to transgene activity using various delivery modalities. Figure 6A shows activity data (expressed as a percentage of normal) for GAA after administration of free AAV1 -CMV-hGAA or gel mediated delivery of AAV1 -CMV-hGAA (compared to mock and wild type controls). Figure 6B shows glycogen staining (representative of clearance of accumulated glycogen) in diaphragm tissue sections after free AAV delivery. Figure 6C shows glycogen staining (representative of clearance of accumulated glycogen) in diaphragm tissue sections after gel-mediated AAV delivery.

[0028] Figure 7 shows data related to diaphragm contractile strength after delivery of AAV1 -CMV- hGAA via different routes.

[0029] Figures 8A-8D show whole-animal ventilatory function data after different treatment scenarios.

[0030] Figure 9 shows a schematic of SARS-CoV-2.

[0031] Figure 10 depicts a schematic of SARS-CoV-2 interaction with the Angiotensin Converting Enzyme-2 receptor.

[0032] Figures 11A-11 B show data related to ELISA assay development. Figure 11 A shows a standard curve generated for an ELISA assay in order to detect antibodies to SARS-CoV-2. Figure 11 B shows the standard data used in the serial dilutions to generate the standard curve. [0033] Figures 12A-12B shows data related to anti-SARS-CoV-2 antibody production. Figure 12 A shows a histogram of patient antibody concentration at the indicated time points after an initial dose of an mRNA-based vaccine and after a booster shot. Figure 12B tabulates the data and includes a control group of COVID-19 positive antibody concentrations.

[0034] Figure 13 shows a correlation curve between patient age at time of vaccination and antibody concentration after the first vaccine dose.

[0035] Figures 14A-14B show data related to anti-SARS-CoV-2 antibody production in rats. Figure 14A shows data related to the concentration of antibodies detected in rats given the indicated treatment. Figure 14B tabulates the data.

DETAILED DESCRIPTION

Definitions

[0036] As used herein the term “nucleic acid” or “oligonucleotide” refers to multiple nucleotides (e.g., molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term includes polynucleosides (i.e. a polynucleotide minus the phosphate) and any other organic base containing polymer. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non- naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. A nucleic acid can include any other suitable modifications. Thus, the term nucleic acid also encompasses nucleic acids with substitutions or modifications, such as in the bases and/or sugars.

[0037] “Exogenous” as used herein with reference to a nucleic acid, e.g., mRNA, has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure. An exogenous nucleic acid generally originates from outside the cell with respect to which the nucleic acid is exogenous. Thus, an exogenous nucleic acid, e.g., exogenous mRNA, is generally not transcribed from the host cell’s genomic DNA in its natural state. In some embodiments, an exogenous nucleic acid, e.g., exogenous mRNA, is not transcribed from the host cell’s DNA. An exogenous mRNA includes a chemically-modified mRNA (CMmRNA) in which one or more bases of the mRNA is chemically modified, as provided herein.

[0038] “Messenger RNA” or “mRNA” refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) or protein and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some embodiments, an mRNA is translated in vivo, e.g., in a host cell. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. [0039] A “3’ untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e. , 3’) from the stop codon (i.e. , the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0040] A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates.

[0041] As used herein, “miRNA” or “miR” refers to small non-coding RNAs, belonging to a class of regulatory molecules found in eukaryotes (e.g., plants and animals) that regulate gene expression by binding to complementary sites (or target sites) on target messenger RNA (mRNA) transcripts. miR are initially expressed in the nucleus as part of longer primary transcripts called primary miRNAs (pri-miRNAs). Inside the nucleus, pri-miRNAs are partially digested by the enzyme Drosha, to form 65-120 nucleotide- long hairpin precursor miRNAs (pre-miRNAs) that are exported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs of 18-25 nucleotides in length, which are the active molecules. In animals, these short RNAs have a 5’ proximal "seed" region (generally nucleotides 2 to 8) which can be the primary determinant of the pairing specificity of the miRNA to the 3’ untranslated region (3’-UTR) of a target mRNA. As used herein, a miR “targets” an mRNA, e.g., an exogenous mRNA, where the miR mediates silencing of expression from the mRNA based on complementarity of the miR seed sequence with a target sequence in the mRNA.

[0042] Polypeptide or nucleic acid molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Any suitable methods and computer programs for the alignment can be used. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981 ) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197.) A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch. C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453.). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0043] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a suitable mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects. Smith. D. W., ed., Academic Press. New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11 -17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al. , Nucleic Acids Research, 12(1 ), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0044] The term “Watson-Crick base-pairing”, or “base-pairing” refers to the formation of hydrogen bonds between specific pairs of nucleotide bases (“complementary base pairs”). For example, two hydrogen bonds form between adenine (A) and uracil (U), and three hydrogen bonds form between guanine (G) and cytosine (C). One method of assessing the strength of bonding between two polynucleotides is by quantifying the percentage of bonds formed between the guanine and cytosine bases of the two polynucleotides (“GC content”). In some embodiments, the GC content of bonding between two nucleic acids of a multimeric molecule (e.g., a multimeric mRNA molecule) is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the GC content of bonding between two nucleic acids of a multimeric molecule (e.g., a multimeric mRNA molecule) is between 10% and 70%, about 20% to about 60%, or about 30% to about 60%. The formation of a nucleic acid duplex via bonding of complementary base pairs can also be referred to as “hybridization”. Generally, two nucleic acids sharing a region of complementarity are capable, under suitable conditions, of hybridizing (e.g., via nucleic acid base pairing) to form a duplex structure. A region of complementarity can vary in size. In some embodiments, a region of complementarity ranges in length from about 2 base pairs to about 100 base pairs. In some embodiments, a region of complementarity ranges in length from about 5 base pairs to about 75 base pairs. In some embodiments, a region of complementarity ranges in length from about 10 base pairs to about 50 base pairs. In some embodiments, a region of complementarity ranges in length from about 20 base pairs to about 30 base pairs.

[0045] “Subject,” as used herein refers to any vertebrate animal, including mammals and non mammals. A subject can include primates, including humans, and non-primate mammals, such as rodents, domestic animals or game animals. Non-primate mammals can include mouse, rat, hamster, rabbit, dog, fox, wolf, cat, horse, cow, pig, sheep, goat, camel, deer, buffalo, bison, etc. Non-mammals can include bird (e.g., chicken, ostrich, emu, pigeon), reptile (e.g., snake, lizard, turtle), amphibian (e.g., frog, salamander), fish (e.g., salmon, cod, pufferfish, tuna), etc. The terms, “individual,” “patient,” and “subject” are used interchangeably herein. [0046] “Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.

[0047] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321 ); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1 - 56081 -569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0048] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation, “e.g.” is used herein to indicate a non- limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The term “about” as used herein to, for example, define the values and ranges of molecular weights means that the indicated values and/or range limits can vary within ±20%, e.g., within ±10%, including within ±5%. The use of “about” before a number includes the number itself. For example, “about 5” provides express support for “5.” Numbers provided in ranges include overlapping ranges and integers in between; for example a range of 1 -4 and 5-7 includes for example, 1 -7, 1 -6, 1 -5, 2-5, 2-7, 4-7, 1 , 2, 3, 4, 5, 6 and 7.

Viral Infections

[0049] Severe Acute Respiratory Syndrome (SARS) is a result of viral infection. The SARS coronavirus (SARS-CoV) was identified in 2003. Coronaviruses are positive sense, single-strand enveloped RNA virus belonging to the family Coronaviridae. The coronavirus nomenclature is derived from the Latin corona, meaning crown. Viral envelope under electron microscopy appears crown-like due to small bulbar projections formed by the viral spike (S) peplomers. SARS-CoV is thought to be an animal virus from an as-yet-uncertain animal reservoir, perhaps bats, that spread to other animals (civet cats) and first infected humans in the Guangdong province of southern China in 2002. Transmission of SARS-CoV is primarily from person to person. It appears to have occurred mainly during the second week of illness, which corresponds to the peak of virus excretion in respiratory secretions and stool, and when cases with severe disease start to deteriorate clinically. Most cases of human-to-human transmission occurred in the health care setting, in the absence of adequate infection control precautions.

[0050] Symptoms are influenza-like and include fever, malaise, myalgia, headache, diarrhea, and shivering (rigors). No individual symptom or cluster of symptoms has proved to be specific for a diagnosis of SARS. Although fever is the most frequently reported symptom, it is sometimes absent on initial measurement, especially in elderly and immunosuppressed patients. Cough (initially dry), shortness of breath, and diarrhea are present in the first and/or second week of illness. Severe cases often evolve rapidly, progressing to respiratory distress and requiring intensive care.

[0051] Recently a new strain, novel coronavirus 2019 (2019-nCoV) now referred to as SARS- CoV-2, infected humans and rapidly spread causing the COVID-19 pandemic. Several vaccines have been developed to combat the SARS-Cov2 virus that causes COVID-19. However, scientists and researchers have found a variant that first arose in South Africa (B.1.351 ) and a variant identified in Brazil (P.1 ) are partially escaping protection provided by these vaccines. In addition, researchers also found a new coronavirus variant (B.1 .526) in New York City and elsewhere in the Northeast that carries mutations that help it evade the body's natural immune response - as well as the effects of monoclonal antibody treatments.

[0052] Patients who are older age, especially > 65 years and people with comorbidities appear more likely to develop an infection and severe symptoms and die. However, early data in the U.S. among hospitalized patients suggest that younger adults are hospitalized also, with adults aged 20-44 accounting for 20% of hospitalizations, and 12% of ICU admissions. Children appear less symptomatic with infection and less prone to severe illness. SARS-CoV-2 is transmitted primarily by respiratory droplets and by deposition of virus on surfaces. Symptoms include, but are not limited to fever (83-98%), cough (46-82%, usually dry), shortness of breath at onset (31%), and/or myalgia or fatigue (11 -44%) and in some patients, one or more of pharyngitis, headache, productive cough, and Gl symptoms. While -80% of infections are not severe and some may be asymptomatic, the asymptomatic nature may facilitate transmission. Infection results primarily in primarily upper and lower respiratory tract infections. For hospitalized patients with pneumonia, initial data suggest that -50% of patients develop hypoxemia by day 8 and Acute Respiratory Distress Syndrome develops in 17-29% of patients. Those patients in the ICU have required non-invasive ventilation (42%) or mechanical ventilation (47%). 11% of ICU patients have required high-flow 02 (11%) while about 2-5% required ECMO. During the initial outbreak in critically ill patients in Washington State in the US, a set of 21 patients were reviewed. 86% had one or more comorbidities. Symptoms in this population lasted for approximately 3.5 days, and admission to the ICU within 24 hours of hospitalization occurred in 81 % of these. 67% of these patients had leukopenia and 71 % required mechanical ventilation. ARDS was observed in 100% of those who required mechanical ventilation, most developing within 72 hours. Cardiomyopathy developed in -33% of patients. The ARDS and cardiomyopathy are results of the direct and indirect effect of the SARs-CoV-2 impact on tissue, discussed in more detail below.

[0053] A vaccine is a biological substance administered to a patient in order to provoke development of acquired immunity to a particular infectious disease. Vaccines often contain an agent that resembles, or is derived from, a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat, destroy it, and to further recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future. Vaccines can be prophylactic (to prevent or ameliorate a future infection) or therapeutic (e.g., vaccines against cancer). [0054] As discussed in more detail below, several embodiments disclosed herein relate to viral vectors encoding a viral protein, or derivative thereof, and administration of these vectors to patients in order to allow their immune systems (through the humoral immune response) to develop antibodies against the viral protein, or derivative thereof. In several embodiments, the viral protein is from MERS-CoV or SARS-CoV. In several embodiments, the viral protein, or derivative thereof is a viral spike protein. In several embodiments, the viral spike protein is from SARS-CoV-2. In several embodiments, the viral protein has been modified, e.g., truncated, mutated, or otherwise been subject to genetic modification in order to improve therapeutic responses in subjects who receive the viral protein, e.g., as a vaccine.

Wild type adeno-associated virus

[0055] Human adeno-associated virus (AAV) is a non-pathogenic parvovirus that only productively replicates in cells co-infected by a helper virus, usually adenovirus or herpes virus. The virus has a wide host range and can productively infect many cell types from a variety of animal species. Sero- epidemiologic studies have shown that most people (50-96%) in the U.S.A. have been exposed to the most common serotype (AAV2), probably as a passenger during a productive adenovirus (Ad) infection. Nevertheless, AAV has not been implicated in any human or animal disease.

[0056] The AAV life cycle is unusual. AAV binds to cells via a heparan sulfate proteoglycan receptor. Once attached, AAV entry is dependent upon the presence of a co-receptor, either the fibroblast growth factor receptor or anb5 integrin molecule. In infected cells, the incoming AAV single-stranded DNA (ssDNA) is converted to double-stranded transcriptional template. Cells infected with AAV and a helper virus will undergo productive replication of AAV prior to cell lysis, which is induced by the helper virus rather than AAV. Helper virus encodes proteins or RNA transcripts which are transcriptional regulators and are involved in DNA replication or that modify the cellular environment in order to permit efficient viral production. Human cells infected with AAV alone, however, become persistently infected. This latency pathway of wild-type AAV often results in site-specific integration on chromosome 19, the AAVS1 site. The AAV genome consists of two 145-nucleotide inverted terminal repeat (TR) sequences, each an identical palindrome at either terminus of the virus, flanking the two AAV open reading frames (orfs), rep and cap. AAV rep and cap genes encode the four Rep proteins (Rep 78, 68, 52 and 40) involved in viral DNA replication, resolution of replicative intermediates and generation of single-strand genomes and the three structural proteins (VP1 , VP2, and VP3) that make up the viral capsid. The two larger rep proteins (Rep 78 and Rep 68) are required for resolution of the AAV termini during productive infections. They are also capable of binding to the human chromosome 19 target sequence for AAV integration and initiating site- specific integration. Thus, rep-deleted recombinant AAV vectors do not integrate site-specifically, but rather persist as a combination of episomal forms and random-site integrants.

Adeno-associated viral vectors [0057] Recombinant AAV (rAAV) vectors are typically produced by replacing the viral coding sequences with transgenes of interest. These vectors have been shown to be highly efficient for gene transfer and expression at a number of different sites in vitro and in vivo. They have consistently mediated stable expression and have been safe in studies performed in the respiratory tract, the central nervous system, skeletal muscle, liver and eye. The efficiency of rAAV-mediated transduction has increased as the titer and purity of rAAV preparations has improved. Skeletal muscle is often chosen as the target tissue because it is accessible, efficiently transduced by rAAV vectors, well vascularized and is able to express and process secreted proteins.

[0058] The TRs from the AAV genome are the only viral sequences required in cis to generate rAAV vectors. Recombinant constructs containing two TRs bracketing a gene expression cassette of ~5 kb are converted into a ssDNA vector genome and packaged into AAV particles in the presence of AAV rep and cap gene products and helper functions, usually from an adenovirus (Ad). Our laboratory has reported technological improvements in both production of and purification of rAAV2.

[0059] Many serotypes of AAV have been cloned and sequenced, with five of the six having divergent amino acid sequences. However, serotypes 1 and 6 share >99% amino acid homology in their capsid proteins. Of the first six AAV serotypes, serotype 2 is best characterized and therefore predominantly used in gene transfer studies, however according to embodiments disclosed herein, other AAV serotypes are used. Comparison of the serotype capsid amino acid sequences suggests that types 1 , 2 and 3 share homology across the three capsids in accord with heparan sulfate binding. Direct intramuscular injections of non-serotype 2 AAV vectors, especially rAAV1 , transduce skeletal muscle more efficiently and secrete canine factor IX at levels two-to-three logs greater than rAAV2. Because the identical transgene cassette was used in the vector constructs, these results suggest that rAAV1 virions are more efficient for gene delivery to muscle. Furthermore, a humoral response was not detected against the transgene protein secreted by intramuscular injection of the rAAV1 construct, contrasting with the significant humoral response elicited by the transgene protein secreted by myocytes transduced by the rAAV2 construct. The lack of cross-reactivity among neutralizing antibodies of different rAAV serotypes suggests that vector readministration using different serotypes of rAAV may be feasible for dose titration. In some embodiments, rAAV1 is used, while other serotypes such as RH-10 and RH-74 are used in additional embodiments.

[0060] In general, there are two different approaches for packaging rAAV vectors: “true type” and “pseudotyped” vectors. The former refers to vectors having TRs, Rep proteins and capsid proteins derived from the same wild-type virus, e.g. AAV2. The latter refers to vectors derived from TRs and Rep proteins of one serotype virus, and capsid proteins of another, e.g. 2 and 1 (AAV2/1 ). The pseudotyping of AAV2- TR-containing vectors was pursued because more experience exists with the safety profile of these TRs in animal models and humans. The chromosomal integration efficiency and specificity has been investigated for AAV2 TRs. [0061] In recent years, there have been significant improvements in production and purification of rAAV vectors. The major improvements in production have included enhanced output of the number of particles per cell and the emergence of a number of scaleable systems. Several groups have independently found that the use of plasmids to express adenovirus (Ad) helper genes in transient transfection results in greater efficiency of rAAV production than infection with Ad virus, perhaps because of enhanced viability of producer cells or the lack of competition with the helper virus for DNA replication machinery. Another interesting finding is that down-regulation of Rep78/68 relative to Rep52/40 and the capsid proteins results in a greater accumulation of single-stranded DNA genomes and packaged vector DNA. The incorporation of these improvements into transient transfection production protocols has enhanced yields from about 1 - 10 IU per cell to over 100 IU per cell. Stable producer cell lines and packaging cell lines used in combination with recombinant hybrid AAV-adenoviruses have achieved 100-300 IU per cell. Hybrid AAV-herpes vectors have achieved outputs that approach the 5,000-10,000 IU per cell seen with wtAAV. Overall, these newer methods produce greater vector yields and reduce or eliminate detectable replication-competent AAV (rcAAV) contamination.

[0062] Early reports comparing the transduction efficiencies and specificities of rAAV vector serotypes relied on CsCI gradients for purification, but this approach can generate vector stocks with large particle:infectious (P:l) ratios. Purification using affinity chromatography, based on identified cellular receptors, is becoming more common and the more physiological conditions result in vector stocks with P:l ratios of <50. An efficient and reproducible protocol based on partial purification of an initial freeze and thaw lysate followed by chromatography for the purification and concentration of rAAV1 vectors has been developed for AAV1 clinical manufacturing. rAAV-based Compositions and Methods for Inducing Humoral Immunity

[0063] Described below are non-limiting embodiments of compositions, such as rAAV-based compositions, and their uses in inducing humoral immune response in subjects in order to prevent or reduce adverse health impacts due to a future infection, such as a viral infection, including COVID-19.

[0064] In several embodiments, a rAAV vector comprises a viral capsid and a nucleic acid vector as described herein, which is encapsidated by the viral capsid. In some embodiments, the nucleic acid vector comprises (1 ) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest, (2) one or more nucleic acid regions comprising a sequence that facilitates expression of the heterologous nucleic acid region (e.g., a promoter and/or enhancer), and (3) one or more nucleic acid regions comprising a sequence that facilitate integration of the heterologous nucleic acid region (optionally with the one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of the subject. In some embodiments, viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences. In some embodiments, the nucleic acid vector comprises one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest operably linked to a promoter, wherein the one or more heterologous nucleic acid regions are flanked on each side with an ITR sequence. The ITR sequences can be derived from any AAV serotype (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, RH-10, RH-74) or can be derived from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2.

[0065] In some embodiments, the nucleic acid vector comprises a pTR-UF-11 plasmid backbone, which is a plasmid that contains AAV2 ITRs. This plasmid is commercially available from the American Type Culture Collection (ATCC MBA-331 ). In several embodiments, the resultant vector is pseudotyped to an alternative sero-type. For example, in several embodiments an AAV2 serotype is pseudotyped to AAV1 .

[0066] In some embodiments, the nucleic acid vector comprises one or more regions comprising a sequence that facilitates expression of the heterologous nucleic acid, e.g., expression control sequences operatively linked to the heterologous nucleic acid. Numerous such sequences are known in the art. Non limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is completed herein (e.g., a promoter and an enhancer).

[0067] To achieve appropriate expression levels of the protein or polypeptide of interest, any of a number of promoters suitable for use in the selected host cell may be employed. The promoter may be, for example, a constitutive promoter, tissue-specific promoter, inducible promoter, or a synthetic promoter. For example, constitutive promoters of different strengths can be used. A nucleic acid vector described herein may include one or more constitutive promoters, such as viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and cytomegalovirus (CMV) promoters. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the b-actin promoter.

[0068] Inducible promoters and/or regulatory elements may also be contemplated for achieving appropriate expression levels of the protein or polypeptide of interest. Non-limiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.

[0069] Tissue-specific promoters and/or regulatory elements are also contemplated herein. Non limiting examples of such promoters that may be used include (1 ) desmin, creatine kinase, myogenin, alpha myosin heavy chain, human brain and natriuretic peptide, specific for muscle cells, and (2) albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters, specific for liver cells.

[0070] Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.

[0071] In some embodiments, a nucleic acid vector described herein may also contain marker or reporter genes, e.g., LacZ or a fluorescent protein. [0072] As a non-limiting embodiment, Figure 3 schematically depicts a rAAV vector encoding a soluble SARS-CoV-2 spike protein. In several embodiments, the nucleic acid sequence of native SARS- CoV-2 spike protein is engineered (e.g., truncated) to remove at least the transmembrane domain. In several embodiments, the modified SARS-CoV-2 spike protein does not comprise SEQ ID NO: 5. In several embodiments, an alternative domain is inserted in place of all or a portion of the native transmembrane domain. By way of example, a disruption element can be used to disrupt the native transmembrane sequence. In such cases, the native transmembrane domain sequence may still be present, but insertions result in a frameshift such that the protein is no longer able to bind/pierce a membrane. For example, in several embodiments, a dimerization or trimerization domain is inserted in order to disrupt the native transmembrane domain or replace the native transmembrane domain. In several embodiments, a trimerization domain is included in the engineered SARS-CoV-2 spike protein. In several embodiments, the trimerization domain encodes a trimerization domain having an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that of SEQ ID NO. 6. This domain replaces (or otherwise disrupts the native transmembrane domain) and allows a soluble trimer of engineered SARS-CoV-2 spike proteins to form, which facilitates circulation of the soluble trimer and induction of a humoral immune response. Additional modifications are made to the engineered SARS- CoV-2 spike protein. In several embodiments, the SARS-CoV-2 spike protein is modified to be optimized (e.g., codon-optimized) for improved expression in humans. In several embodiments, stabilizing modifications are introduced into the engineered SARS-CoV-2 spike protein. For example, in several embodiments a K985P and/or a V986P mutation are introduced. In several embodiments, stabilizing modifications are made in conjunction with a trimerization domain being inserted in place of the native transmembrane domain. In several embodiments, the engineered SARS-CoV-2 spike protein is encoded by a nucleic acid sequence that encodes a spike protein having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that of SEQ ID NO. 4. In several embodiments, the engineered SARS-CoV-2 spike protein comprises SEQ ID NO. 4. In some embodiments, the nucleic acid vector (e.g., a rAAV vector) comprises one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest. The nucleic acid vector, according to some embodiments comprises a vector having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that of SEQ ID NO. 2. The nucleic acid vector, according to some embodiments comprises a vector having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that of SEQ ID NO. 1 .

[0073] In several embodiments, the engineered SARS-CoV-2 spike protein is encoded by a nucleic acid sequence that encodes a spike protein derived from SEQ ID NO. 3. In several embodiments, the engineered SARS-CoV-2 spike protein is encoded by a nucleic acid sequence that encodes a spike protein having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that of SEQ ID NO. 7, 8, or 9. In several embodiments, the nucleic acid sequence of native SARS- CoV-2 spike protein is engineered (e.g., truncated) to yield 1 , 2, 3, 4, or more epitopes, such as for example, highly antigenic sequences. In several embodiments, those highly antigenic sequences are separated by spacer nucleic acid, in order to allow each of the plurality of epitopes to be processed.

[0074] In several embodiments, administration of a nucleic acid vector is by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to a target tissue. In several embodiments, the dosing is intramuscular invention. In several embodiments, a plurality of injections, or other administration types, are provided, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more injections. In several embodiments, management of potential immune response to the viral can be undertaken through methods, such as those disclosed in United States Patent Application No. 15/306,139, which is incorporated by reference herein in its entirety.

[0075] Depending on the embodiment, the dose of a first rAAV vector can be between about 1 X 10¹⁰ vector genomes (VG) and about 1 x 10¹⁴ VG, including about 5 X 10¹⁰ VG, 1 X 10¹¹ , 5 X 10¹¹, 1 X 10¹², 5 X 10¹², 1 X 10¹³, 5 X 10¹³, 1 X 10¹⁴ and any dose in between or inclusive of those doses listed. When used, a first and second rAAV can be dosed in relation to one another, or based on the target tissue for expression. For example the ratio of the first rAAV to the second rAAV can be about 1 :1 , 1 :2, 1 :4, 1 :5, 1 :7, 1 :10, 1 :20, 1 :50, 1 :100, 1 :200, 1 :500, 500:1 , 200:1 , 100:1 , 50:1 , 20:1 , 10:1 , 7:1 , 5:1 , 4:1 , 2:1 , or any ratio in between or inclusive of those ratios listed. In several embodiments, depending on the subject to be treated, the dose is on a vector genome/kilogram body mass basis.

[0076] In several embodiments, the rAAV particles described herein are administered to otherwise healthy individuals in order to induce development of a humoral immune response against the transgene encoded by the rAAV vector, such as the SARS-CoV-2 truncated spike protein (such as an engineered protein with at least about 95% sequence identity to SEQ ID NO: 4). The rationale for this approach is based on prior studies with rAAV particles.

[0077] rAAV vectors can be used to safely deliver and achieve long lasting expression of the encoded transgene. Recently, research teams have been working toward a treatment of Pompe disease based on delivery of adeno-associated vectors, with promising data resulting AAV serotypes 1 and 9. These data are in keeping with that from other laboratories, which have also shown enhanced tropism of AAV 1 and 9 for skeletal muscle. As shown in Figure 4, direct diaphragmatic transduction in a mouse model of muscular dystrophy showed higher levels of transgene expression resulting from AAV1 vectors as compared to serotypes 2 and 5. Furthermore, as shown in Figure 5, AAV9 gave rise to similar levels of diaphragmatic transduction as compared to AAV1 .

[0078] Direct diaphragmatic transduction in mouse models can be achieved via intravenous or intrathoracic injection; direct application or injection, or via gel-mediated delivery. As shown in Figure 6 a gel-mediated method of delivery resulted in significantly enhanced levels of transduction of diaphragm tissue as compared to direct application. Using a mouse model of Pompe disease, supranormal levels of GAA enzyme activity after gel-mediated delivery of AAV1 -CMV-hGAA were achieved. Furthermore, the high levels of GAA activity resulted in the clearance of accumulated glycogen in the diaphragm tissue, as determined by periodic acid-Schiff (PAS) staining (Figure 6). In addition to the biochemical and histological correction effected by AAV1 -mediated gene transfer, significant physiological correction of diaphragm muscle function and ventilation was achieved.

[0079] As shown in Figure 7, gel-mediated delivery of AAV1 -CMV-hGAA to adult animals (3 and 9 months of age) led to significantly improved diaphragm muscle contractile strength at 1 year of age (and therefore 9 and 3 months post-vector delivery, respectively) as compared to age-matched, untreated controls. As shown in Figure 8, the improvement in diaphragm muscle function correlated with significantly improved ventilatory capabilities. Using whole-body barometric plethysmography, we are able to assay ventilatory function in unrestrained, awake animals. Animals that were treated at 3, 9 and 21 months of age all showed significantly improved minute ventilations.

[0080] The potential for intravenous administration of AAV1 -CMV-hGAA to correct diaphragm was also investigated. As shown in Figure 8, long-term (11 months post-injection) therapeutic levels of GAA activity were achieved after a single intravenous administration of vector to Pompe disease mouse neonates. In addition to the sustained levels of therapeutic enzyme activity, a dramatic improvement in the diaphragm function was demonstrated, as determined by measurement of diaphragm contractile force (Figure 7) as well as significant improvement in multiple parameters of ventilatory function including frequency, tidal volume, minute ventilation and peak inspiratory flow (Figure 8). These data collectively demonstrate that rAAV vectors can be used to safely deliver and achieve long lasting expression of the encoded transgene, together with associated physiological changes.

[0081] To date, only six rAAV vectors have been tested in humans: rAAV-CFTR, rAAV-factor IX, rAAV-sarcoglycan, rAAV-aspartoacylase, rAAV-alpha-1 antitrypsin and rAAV-microdystrophin. Extensive work has been done on the rAAV-factor IX vector by a consortium of investigators from the Children’s Hospital of Pennsylvania, Stanford University and Avigen. The rAAV-factor IX vector was shown to be capable of long-term correction of the coagulopathy in both the factor IX-deficient mouse and the hemophilia B dog model. Intramuscular administration and portal vein administration were both efficacious in the dog model. Intramuscular administration in the mouse model was associated with the development of a humoral immune response to factor IX, which appears to have been related to the adherence of factor IX to type IV collagen in the extracellular matrix of the muscle. A clinical trial of intramuscular administration was reported, in which some biological activity of the vector was noted at a low dose, without obvious toxicity. The trial for Canavan’s disease (aspartoacyclase deficiency) has been completed without adverse events. Currently, the trial of AAV-1 expressing alph-1 antitrypsin has been completed at our institution, with no adverse events reported. Additionally, our center has completed enrollment of ten subjects in Cohorts l-IV of the “Phase I Trial of Ocular Sub-Retinal Injection of a Recombinant Adeno-Associated Virus (rAAV- RPE65) Gene Vector in Patients with Retinal Disease Due to RPE65 Mutations.” There are many similarities in the RPE65 study to the study and technology here, where a surgical route of delivery is used to reach the target tissue. There is evidence of safety at two dose levels as well as indication of restoration of retinal function and improved vision in that study. [0082] To that end, and in view of the embodiments disclosed herein, a clinical study will be performed to verify the safety and efficacy of certain embodiments disclosed herein. The study will be conducted in healthy control adult subjects at two dosing levels, 5 x 10¹² vg and 1 x 10¹³ vg delivered intramuscularly in the quadriceps muscle. This is a first-in-human, phase I/ll study to evaluate the safety, tolerability, and efficacy of AAV1 -CMV-Sol_ Spike (rAAV1 encoding a transmembrane-region deleted SARS-CoV-2 spike protein) as a vaccine to combat the pandemic outbreak of SARS-CoV-2 causing COVID-19. This study will test several important concepts leading to immediate immunization of susceptible individuals. The critical advantage of an AAV-based vaccine is that single administration in a 1.0 ml IM injection will lead to ongoing expression of the modified soluble coronavirus spike protein and elicit a potent anti-spike polyclonal response. Based on non-clinical studies, the level of antibody should exceed the threshold for effective neutralization of SARS2.

[0083] In certain embodiments, treatment of a subject with a rAAV particles as described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0084] The subject treated by the present methods can be any suitable subject in need of treatment of a viral infection. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the subject has one or more comorbidities, such as, but not limited to a cardiorespiratory dysfunction, hypertension, diabetes, and/or coronary heart disease. “Subject” and “patient” are used interchangeably herein. In several embodiments, the subject may have already been exposed to a given virus, or variant thereof, previously.

[0085] In some embodiments, rAAV particles as disclosed herein are administered prior to a later administered therapeutic agent. In several embodiments, the later administered therapeutic agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a corticosteroid, such as, without limitation, cortisone hydrocortisone, hydrocortisone-21 -monoesters (e.g., hydrocortisone-21 - acetate, hydrocortisone-21 -butyrate, hydrocortisone-21 -propionate, hydrocortisone-21 -valerate, etc.), hydrocortisone-17,21 -diesters (e.g., hydrocortisone-17,21 -diacetate, hydrocortisone-17-acetate-21 - butyrate, hydrocortisone-17,21 -dibutyrate, etc.), alclometasone, dexamethasone, flumethasone, prednisolone, methylprednisolone, betamethasone, typically as betamethasone benzoate or betamethasone diproprionate; fluocinonide; prednisone; and triamcinolone, typically as triamcinolone acetonide. In some embodiments, the anti-inflammatory agent is a mast cell degranulation inhibitor, such as, without limitation, cromolyn (5,5'-(2-hydroxypropane-1 ,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2- carboxylic acid) (also known as cromoglycate), and 2-carboxylatochromon-5'-yl-2-hydroxypropane derivatives such as bis(acetoxymethyl), disodium cromoglycate, nedocromil (9-ethyl-4,6-dioxo-10-propyl- 6,9-dihydro-4H-pyrano[3,2-g]quinoline-2,8-dicarboxylic acid) and tranilast (2-{[(2E)-3-(3,4- dimethoxyphenyl)prop-2-enoyl]amino}), and lodoxamide (2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2- oxoacetic acid). In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drugs (NSAIDs), such as, without limitation, aspirin compounds (acetylsalicylates), non-aspirin salicylates, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, naproxen sodium, phenylbutazone, sulindac, and tometin.

[0086] In some embodiments, the anti-inflammatory agent is an antihistamine, such as, without limitation, clemastine, clemastine fumarate (2(R)-[2-[1 -(4-Chlorophenyl)-1 -phenyl-ethoxy]ethyl-1 - methylpyrrolidine), dexmedetomidine, doxylamine, loratidine, desloratidine and promethazine, and diphenhydramine, or pharmaceutically acceptable salts, solvates or esters thereof. In some embodiments, the antihistamine includes, without limitation, azatadine, azelastine, burfroline, cetirizine, cyproheptadine, doxantrozole, etodroxizine, forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil, N,N'- substituted piperazines or terfenadine. In some embodiments, the antihistamine is an H1 antagonist, such as, but not limited to, cetirizine, chlorpheniramine, dimenhydrinate, diphenhydramine, fexofenadine, hydroxyzine, orphenadrine, pheniramine, and doxylamine. In some embodiments, the antihistamine is an H2 antagonist, such as, but not limited to, cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine.

[0087] In some embodiments, the therapeutic agent is an antiviral agent, including antiretroviral agents. Suitable antiviral agents include, without limitation, acyclovir, famcyclovir, ganciclovir, foscarnet, idoxuridine, sorivudine, trifluorothymidine, valacyclovir, vidarabine, didanosine, dideoxyinosine, stavudine, zalcitabine, zidovudine, amantadine, interferon alpha, ribavirin and rimantadine.

[0088] In some embodiments, the therapeutic agent is an antibiotic. Non-limiting examples of suitable antibiotics include beta-lactams such as penicillins, aminopenicillins (e.g., amoxicillin, ampicillin, hetacillin, etc.), penicillinase resistant antibiotics (e.g., cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, etc.), extended spectrum antibiotics (e.g., axlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, etc.); cephalosporins (e.g., cefadroxil, cefazolin, cephalixin, cephalothin, cephapirin, cephradine, cefaclor, cefacmandole, cefmetazole, cefonicid, ceforanide, cefotetan, cefoxitin, cefprozil, cefuroxime, loracarbef, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftiofur, ceftizoxime, ceftriaxone, moxalactam, etc.); monobactams such as aztreonam; Carbapenems such as imipenem and eropenem; quinolones (e.g., ciprofloxacin, enrofloxacin, difloxacin, orbifloxacin, marbofloxacin, etc.); chloramphenicols (e.g., chloramphenicol, thiamphenicol, florfenicol, etc.); tetracyclines (e.g., chlortetracycline, tetracycline, oxytetracycline, doxycycline, minocycline, etc.); macrolides (e.g., erythromycin, tylosin, tlimicosin, clarithromycin, azithromycin, etc.); lincosamides (e.g., lincomycin, clindamycin, etc.); aminoglycosides (e.g., gentamicin, amikacin, kanamycin, apramycin, tobramycin, neomycin, dihydrostreptomycin, paromomycin, etc.); sulfonamides (e.g., sulfadmethoxine, sfulfamethazine, sulfaquinoxaline, sulfamerazine, sulfathiazole, sulfasalazine, sulfadiazine, sulfabromomethazine, suflaethoxypyridazine, etc.); glycopeptides (e.g., vancomycin, teicoplanin, ramoplanin, and decaplanin; and other antibiotics (e.g., rifampin, nitrofuran, virginiamycin, polymyxins, tobramycin, etc.).

[0089] In some embodiments, the therapeutic agent is an antifungal agent, such as, but not limited to, itraconazole, ketoconazole, fluoconazole, and amphotericin B. In some embodiments, the therapeutic agent is an antiparasitic agents, such as, but not limited to, the broad spectrum antiparasitic medicament nitazoxanide; antimalarial drugs and other antiprotozoal agents (e.g., artemisins, mefloquine, lumefantrine, tinidazole, and miltefosine); anthelminthics such as mebendazole, thiabendazole, and ivermectin; and antiamoebic agents such as rifampin and amphotericin B.

[0090] In some embodiments, the therapeutic agent is an analgesic agent, including, without limitation, opioid analgesics such as alfentanil, buprenorphine, butorphanol, codeine, drocode, fentanyl, hydrocodone, hydromorphone, levorphanol, meperidine, methadone, morphine, nalbuphine, oxycodone, oxymorphone, pentazocine, propoxyphene, sufentanil, and tramadol; and nonopioid analgesics such as apazone, etodolac, diphenpyramide, indomethacin, meclofenamate, mefenamic acid, oxaprozin, phenylbutazone, piroxicam, and tolmetin.

[0091] The methods of the present disclosure can enhance the humoral immune response to a variety of pathogens. Pathogens include, without limitation, one or more of the following: viruses (including but not limited to coronavirus, human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza virus, hepatitis virus, Coxsackie Virus, herpes zoster virus, measles virus, mumps virus, rubella, rabies virus, hemorrhagic viral fevers, H1 N1 , and the like), prions, parasites, fungi, mold, yeast and bacteria (both gram-positive and gram-negative). In some embodiments, pathogens include, without limitation, Candida albicans, Aspergillus niger, Escherichia coli (E. coli), Pseudomonas aeruginosa ( P . aeruginosa), and Staphylococcus aureus (S. aureus), Group A streptococci, S. pneumoniae, Mycobacterium tuberculosis, Campylobacter jejuni, Salmonella, Shigella, and a variety of drug resistant bacteria.

[0092] In some embodiments, the pathogen is a virus, e.g., a DNA or RNA virus. In some embodiments, the virus is an RNA virus, e.g., a single or double-stranded virus. In some embodiments, the RNA virus is a positive sense, single-stranded RNA virus. In some embodiments, the virus belongs to the Nidovirales order. In some embodiments, the virus belongs to the Coronaviridae family. In some embodiments, the virus belongs to the alphacoronavirus, betacoronavirus, gammacoronavirus or deltacoronavirus genus. In some embodiments, the alphacoronavirus is, without limitation, human coronavirus 229E, human coronavirus NL63 or transmissible gastroenteritis virus (TGEV). In some embodiments, the betacoronavirus is, without limitation, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), SARS-CoV-2 (COVID-19), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), human coronavirus HKU1 , or human coronavirus OC43. In some embodiments, the gammacoronavirus is infectious bronchitis virus (IBV). EXAMPLES

[0093] The following Examples (including prophetic examples) are non-limiting examples of methods and materials that can be used according to embodiments disclosed herein.

Clinical Evaluation

[0094] Assessment of the safety of intramuscular administration of a recombinant adeno- associated virus vector, rAAV1 -CMV-SARS2, in healthy adult volunteers. Two dose levels (1.0 x 10¹³ and 5.0 x 10¹² vg) will be studied. One goal is to determine the safety of rAAV1 -CMV-SARS2 vector delivered intramuscularly in healthy adults as well as to determine the efficacy of rAAV1 -CMV-SARS2 vector in producing the anti-SARS antibody. To accomplish these goals, a sequential two-arm, phase I/ll study evaluating the safety and potential efficacy of a single administration of rAAV1 -CMV-SARS2 vector injected intramuscularly into the quadricep for the purpose of anti-SARS-CoV-2 antibody production is described below. Three subjects per dose level are planned. Each arm of 3-4 subjects will be enrolled sequentially and enrollment into the next higher dose level will be dependent on assessment of dose-limiting toxicity.

[0095] The study population will be made up of 6 subjects, male or female, 18-75 years of age, three with an anti-AAV1 titer of <100U/ml and three with an anti-AAV1 titer of 100-500 U/ml. All subjects will be healthy volunteers who test negative for SARS-CoV-2 at screening.

[0096] Following screening visit and vaccine administration at day 0, subjects will be evaluated at a clinical site on days 3, 7, 14, 28 and 90, of the trial. Following day 90, subjects will be evaluated by the study nurse by telephone, or video conference when available. Subjects may be provided with a sample collection kit if they are experiencing any long-term side-effects. Blood samples will be collected at an outpatient facility and shipped to the clinical site for analysis. At each visit to the clinical site, subjects will have a physical examination and laboratory evaluation of chemistry and hematology.

[0097] In addition, at days 3, 7, 14, 28 and 90 the presence of vector in peripheral blood will be evaluated. Testing for the presence of anti-AAV and anti-SARS-CoV-2 antibodies will occur at screening, day 0 (pre-injection), and days 3, 7, 14, 28 and 90.

[0098] Safety will be assessed by measurement of changes in serum chemistries, coagulation and hematology, urinalysis and immunologic response to SARS-CoV-2 and AAV as well as reported subject history of any symptoms.

[0099] Impact of the vaccine in promoting the production of anti-SARS-CoV-2 antibodies will be assessed by measurement of IgG and IgM for SARS2 in the blood. Testing will be done at screening, baseline and 3, 7, 14, 28 and 90 days following vaccine administration.

Development of ELISA Assay for Detection of SARS-Cov-2

[00100] In order to assess the efficacy of the AAV-based therapies disclosed herein, an ELISA- based assay was developed. As discussed above, the SARS-CoV-2 virus is a single stranded RNA virus and comprises a viral envelope protein, an outer membrane, a nucleocapsid and a spike protein. This is schematically shown in Figure 9. Functionally, the envelope protein is known to be involved in viral assembly. The viral membrane promotes spike incorporation and also facilitates production of additional virions. The nucleocapsid aids in viral entry into a host cell and additional functions post-entry. Finally, the spike protein is made up of two components, S1 and S2 domains. The S1 domain facilitates attachment of the virus to its target on host cells, Angiotensin Converting Enzyme-2 (ACE-2, shown schematically in Figure 10). The S2 domain promotes membrane fusion with the host cell. Viral therapies such as those disclosed herein would aid in preventing or reducing the effects of COVID infection by allowing a host to produce antibodies against one or more antigens from SARS-CoV-2. In several embodiments, one or more portions of the spike protein are used in the vaccine.

[00101] A sandwich ELISA assay was developed in order to measure the amount of antibody production in response to a vaccine as compared to a natural infection with SAFtS-Cov-2. A commercially available SARA-CoV-2 S1 spike protein fragment (YP_009724390, Val16-Arg685 from Sino Biological (Catalog No. 40591 -V08H) was coated on wells of a 96-well ELISA plate (2 ug/mL) and incubated overnight at 4 degrees C. Wells were washed 4 times with 300 uL of 0.05% PBS-Tween 20. Wells were then blocked with 3% non-fat milk in 0.05% PBS Tween 20 (incubated for 2 hours at 37 degrees C). Standards and unknowns were prepared in 1% non-fat milk in 0.05% PBS-Tween 20. Wells were washed 4 times with 300 uL of 0.05% PBS-Tween 20 and 100 uL of standards and unknown samples were added to corresponding wells and incubated at 37 degrees C for 2 hours. Standards were generated using serial dilutions of Creative Biolabs Anti-SARS-CoV S therapeutic antibody (CR3022) which was obtained from a person who survived a bout of severe acute respiratory syndrome (SARS), which, as discussed above, is closely related to SARS-CoV-2.

[00102] Wells were washed 4 times with 300 uL of 0.05% PBS-Tween 20 and 100 uL of diluted goat anti-human IgG secondary antibody was added (1 :3000 dilution in 1% milk in 0.05% PBS Tween 20, horse radish peroxidase conjugated) and incubated for 1 hour at 37 degrees C. Wells were washed 4 times with 300 uL of 0.05% PBS-Tween 20. 100 uL of TMB (3,3', 5,5”-tetramethylbenzidine) was added to each well and incubated in the dark for 6 minutes at room temperature. The reaction was stopped with addition of 100 uL of 1 molar phosphoric acid and optical density was measured (450 nm).

[00103] Figure 11 A shows the graphical results of the standard curve that was generated and Figure 11 B tabulates that data. The R^A2 value of 0.999 demonstrates the accuracy of the standard curve for detecting antibody concentrations.

Detection of SARS-Cov-2 Antibodies in Patients After Treatment

[00104] Patients were administered one of two available mRNA-based vaccines (either the Pfizer/BioNTech or Moderna) at time zero in the study and again at 4 weeks per the FDA approved process. Patient antibody levels were evaluated at the indicated points after administration. Figure 12A shows a histogram of detected antibody concentrations. As shown, antibodies are detectable as early as three weeks after the first injection and are still significantly elevated at 8 weeks. Figure 12B shows tabulated data for the experiment blocked by time and in comparison to a COVID-19 positive patient. These data show that these vaccines resulted in antibody concentrations nearly equal to those of a pool of four diagnosed COVID-19 patients after a single dose of the vaccine and that the ELISA methodology described herein is able to detect the resultant antibodies generated by them patients. It is believed that, according to several embodiments, the compositions viral vector-based vaccines disclosed herein will be successful at inducing generation of clinically relevant quantities of antibodies against SARS-CoV-2 and are therefore useful in developing immunity to the virus.

[00105] Figure 13 shows a correlation between the age of the subjects at the time of the first dose of the vaccine and antibody titer after the first dose. Advantageously, as shown by the low R^A2 value, successfully producing antibodies is independent of age of the subject. Thus, according to several embodiments, a SAFtS-CoV-2 vaccine, such as the viral vector-based vaccines disclosed herein are believed to be useful across a wide range of patient age groups.

Dose-Dependent Antibody Production Induced by Vaccine

[00106] Further studying the ability of the vaccines disclosed herein to induce meaningful antibody production post-administration, a dosing, vaccine design, and route of administration study was performed. Rats were divided into sham vs. treatment groups. Treatment groups included those to receive: (i) an injection of 5 x 10¹¹ AAV vector genomes of AAV1 -CMV-SARS-CoV-2 encoding a truncated spike protein that lacks the transmembrane domain (SARS2_01 ) such that the spike protein fragment will be generated by the rats in a soluble format; (ii) an injection of 5 x 10¹¹ AAV vector genomes of AAVI -CMV-SARS-C0V- 2 encoding a truncated spike protein that includes the transmembrane domain (SARS2_02) such that the spike protein fragment will be expressed by rat cells in a membrane-tethered form, or (iii) sublingual administration of 5 x 10¹² AAV vector genomes of AAVI -CMV-SARS-CoV-2 encoding a truncated spike protein that lacks the transmembrane domain (SARS2_01 ) such that the spike protein fragment will be generated by the rats in a soluble format.

[00107] Figure 14A shows the concentration of rat anti-S1 IgG antibodies over four weeks post injection (or sublingual administration). As expected sham animals produced no antibodies, as was also the case with the sublingual gel delivery, despite the higher viral dose. The data for the two injected treatment groups show a time-dependent increase in antibody concentration, with the levels still increasing, even at four weeks post-injection. The membrane-tethered format (SARS2_02) induced modestly increased antibody production. These data demonstrate that, according to several embodiments, delivery of a vector encoding at least a portion of the SARS-CoV-2 spike protein, whether soluble or membrane- tethered, results in production of antibodies against the spike protein in significant amounts, thereby demonstrating use of constructs disclosed herein as vaccines to prevent or reduce symptoms of COVID- 19 infection. In several embodiments, use of the compositions disclosed herein are effective vaccines, even in the face of lack of conservation of the sequence encoding the spike protein (e.g., against COVID- 19 variants). [00108] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00109] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%. ” In some embodiments, at least 95% homologous includes 96%, 97%, 98%, 99%, and 100% homologous to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises”, “consists of” or “consists essentially of” the recited sequence.

[00110] In several embodiments, there are provided amino acid sequences that correspond to any of the nucleic acids disclosed herein, while accounting for degeneracy of the nucleic acid code. Furthermore, those sequences (whether nucleic acid or amino acid) that vary from those expressly disclosed herein, but have functional similarity or equivalency are also contemplated within the scope of the present disclosure. The foregoing includes mutants, truncations, substitutions, or other types of modifications.

[00111] Any titles or subheadings used herein are for organization purposes and should not be used to limit the scope of embodiments disclosed herein.

Claims

WHAT IS CLAIMED IS:

1 . A method for inducing an immune response in a host comprising administering to the host an effective amount of an infectious, recombinant adeno-associated virus (rAVV) encoding at least a portion of a coronavirus spike glycoprotein, wherein the at least a portion of the coronavirus spike glycoprotein does not comprises a complete transmembrane domain, and wherein the immune response induced is an humoral immune response to the at least a portion of the coronavirus spike glycoprotein.

2. The method of Claim 1 , wherein the at least a portion of the coronavirus spike glycoprotein is at least a portion of a SARS-Cov-2 polypeptide.

3. The method of Claim 2, wherein the at least a portion of the coronavirus spike glycoprotein comprises an S1 subunit of the SARS-Cov-2 spike glycoprotein.

4. The method of Claim 2, wherein the at least a portion of the coronavirus spike glycoprotein comprises an N-terminal domain of an S1 subunit of the SARS-Cov-2 spike glycoprotein.

5. The method of Claim 2, wherein the at least a portion of the coronavirus spike glycoprotein comprises an C-terminal domain of an S1 subunit of the SARS-Cov-2 spike glycoprotein.

6. The method of Claim 2, wherein the at least a portion of the coronavirus spike glycoprotein comprises an S2 subunit of the SARS-Cov-2 spike glycoprotein.

7. The method of Claim 1 , wherein the at least a portion of the coronavirus spike glycoprotein is at least a portion of a MERS-Cov-2 polypeptide.

8. The method of Claim 1 , wherein the at least a portion of the coronavirus spike glycoprotein is at least a portion of a SARS-CoV polypeptide.

9. The method of any one of Claims 1 to 8, wherein the lack of a complete transmembrane domain results in expression of the at least a portion of the coronavirus spike glycoprotein as a soluble protein.

10. The method of Claim 1 , wherein the at least a portion of the coronavirus spike glycoprotein does not comprise SEQ ID NO: 5.

11 . The method of any one of Claims 1 to 10, wherein the at least a portion of the coronavirus spike glycoprotein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6.

12. The method of any one of Claims 1 to 11 , wherein the inclusion of the sequence at least 95% homologous to SEQ ID NO: 6 allows the at least a portion of the coronavirus spike glycoprotein to be expressed as a soluble trimer.

13. The method of any one of Claims 1 to 12, wherein the portion of the coronavirus spike glycoprotein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 4.

14. The method of any one of Claims 1 to 12, wherein the portion of the coronavirus spike glycoprotein comprises a sequence having at least 97% sequence identity to SEQ ID NO: 4.

15. The method of any one of Claims 1 to 12, wherein the portion of the coronavirus spike glycoprotein comprises a sequence having at least 99% sequence identity to SEQ ID NO: 4.

16. The method according to any one of Claims 1 to 15, wherein the rAAV has a sequence sharing at least 85% sequence identity to SEQ ID NO: 2.

17. The method according to Claim 16, wherein the rAAV has a sequence sharing at least 95% sequence identity to SEQ ID NO: 2.

18. The method Claim 1 , wherein the portion of the coronavirus spike glycoprotein comprises a sequence at least 95% homologous to one or more of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

19. The method of any one of Claims 1 to 18, wherein the rAAV is a serotype 1 , rh10, rh74 or is pseudotyped to serotype 1 , rh10, rh74.

20. The method of any one of Claims 1 to 19, wherein the rAAV comprises a promoter to drive expression of the at least a portion of the coronavirus spike glycoprotein.

21 . The method of Claim 20, wherein the promoter is a cytomegalovirus promoter.

22. The method of any one of Claims 1 to 19, wherein the administration of the rAAV results in induction of a humoral immune response against the at least a portion of the coronavirus spike glycoprotein.

23. The method according to any one of Claims 1 to 22, wherein the rAAV is produced by a mammalian host cell stably transfected with a recombinant adeno-associated virus genome lacking adeno- associated virus rep-cap genes and stably transfected with adeno-associated virus rep-cap genes.

24. The method according to any one of Claims 1 to 23 wherein the rAAV encoding at least a portion of a coronavirus spike glycoprotein is administered by intramuscular injection.

25. A polynucleotide encoding a first nucleic acid vector containing a first heterologous nucleic acid region encoding a first protein or polypeptide and nucleic acid regions comprising an inverted terminal repeat (ITR) flanking each side of the heterologous nucleic acid region, wherein the heterologous nucleic acid region encodes at least a portion of a coronavirus spike glycoprotein, but does not encode a functional transmembrane domain.

26. The polynucleotide of Claim 25, wherein the lack of a functional transmembrane domain results in expression of the at least a portion of the coronavirus spike glycoprotein as a soluble protein.

27. The polynucleotide of Claim 25 or 26, wherein the polynucleotide encodes a rAAV serotype 1 , rh10, or rh74 vector.

28. The polynucleotide according to any one of Claims 24 to 27, wherein the polynucleotide does not encode SEQ ID NO: 5.

29. The polynucleotide according to any one of Claims 24 to 28, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6.

30. The polynucleotide according to any one of Claims 24 to 29, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4.

31 . The polynucleotide according to any one of Claims 24 to 30, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4.

32. The polynucleotide according to any one of Claims 24 to 31 , wherein the polynucleotide encodes a rAAV with a sequence sharing at least 85% sequence identity with SEQ ID NO: 2.

33. Use of the polynucleotide according to any one of Claims 24 to 31 , for inducing immunity to a coronavirus infection.

34. Use of the polynucleotide according to any one of Claims 24 to 31 , for the preparation of a medicament for inducing immunity to a coronavirus infection.

35. The use of Claim 33 or 34, wherein the coronavirus is SARS-CoV-2.